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www.nature.com/npjbcancer ARTICLE OPEN Effects of neoadjuvant trastuzumab, pertuzumab and palbociclib on Ki67 in HER2 and ER-positive breast cancer 1✉ 2 3 4 5 6,7 8 Luca Gianni , Marco Colleoni , Giancarlo Bisagni , Mauro Mansutti , Claudio Zamagni , Lucia Del Mastro , Stefania Zambelli , 8 9 1 1 10 Giampaolo Bianchini , Antonio Frassoldati , Ilaria Maffeis , Pinuccia Valagussa and Giuseppe Viale The crosstalk between estrogen and HER2 receptors and cell-cycle regulation sustains resistance to endocrine therapy of HER2- and hormone receptor-positive breast cancer. We earlier reported that women with HER2 and ER-positive breast cancer receiving neoadjuvant dual HER2-block and palbociclib in the NA-PHER2 trial had Ki67 decrease and 27% pathological complete responses (pCR). We extended NA-PHER2 to Cohort B using dual HER2-block and palbociclib without fulvestrant and report here Ki67 drops at week-2 (mean change −25.7), at surgery (after 16 weeks, mean change −9.5), high objective response (88.5%) and pCR (19.2%). In Cohort C [Ki67 > 20% and HER2 (IHC 1+/2+ without gene ampliﬁcation)], women also low received fulvestrant, had dramatic Ki67 drop at week 2 (−29.5) persisting at surgery (−19.3), and objective responses in 78.3%. In view of the favorable tolerability and of the efﬁcacy-predictive value of Ki67 drop at week-2, the chemotherapy-free approach of NA-PHER2 deserves further investigation in HER2 and ER-positive breast cancer. The trial is registered with ClinicalTrials.gov, number NCT02530424. npj Breast Cancer (2022) 8:1 ; https://doi.org/10.1038/s41523-021-00377-8 INTRODUCTION RESULTS It has been experimentally established that signaling pathways Patients through the estrogen (ER) and the erbB2 (HER2) receptors converge Results for cohort A were already published . A summary of 1–3 on CDK1 and eventually to Rb checkpoint regulation .We, patients’ disposition for cohorts B and C is outlined in Fig. 1. therefore, postulated that concomitant triple block of ER, HER2, and Overall, 39 patients were registered into cohort B between Rb would result in enhanced antitumor activity that is especially December 20, 2016, and October 27, 2017, and all received at attractive in ER+ and HER2+ breast carcinomas. In those now so least one cycle of planned therapy. The positive HER2 status was called “triple-positive” tumors neoadjuvant trials consistently not conﬁrmed in 6 cases, 2 additional cases had ER status ≤ 10%, showed a lower rate of pathologic complete response (pCR) than mandatory biopsy was unavailable in 4, and one patient received in ER negative HER2+ tumors upon exposure to HER2-directed another systemic therapy in the absence of disease progression. 4–6 therapies in combination with chemotherapy . In the NA-PHER2 A total of 26 patients represent the eligible population of the trial we initially tested the effect of the concomitant triple block of study. Of the eligible patients, 25 had invasive cancer cells in the ER, HER2, and Rb on Ki67 labeling index and found a very rapid tissue samples collected at week 2 after beginning study therapy, and signiﬁcant decrease of proliferation at week 2, a clinical and 21 had invasive cancer cells in tissue samples collected at response rate in more than 95% of patients by week 16 of therapy, surgery. Table 1 describes the main patient characteristics of the and a rate of pCR of 27% at surgery . eligible population. Of note, about 19% of the patients had In view of the good and almost preferential antitumor activity of clinical T3 or T4 and more than 50% had clinically palpable palbociclib monotherapy in HER2+ cell lines , a dual block of axillary lymph nodes. HER2 together with a block of RB1 may be adequate even without A total of 28 patients were registered into cohort C between targeting the estrogen receptor. In addition, HER2 functional May 5, 2017, and February 16, 2018, and all received at least one activation is a well-known mechanism of endocrine resistance in cycle of planned therapy. HER2 status was not conﬁrmed in 1 case, ER+/HER2 negative breast cancer . We, therefore, expanded the 2 additional cases had negative PgR status, in one case Ki67 value NA-PHER2 study to two additional cohorts of patients. In cohort B was <20%, and surgical biopsy was unavailable in one case. A total we explored the effects of a concomitant block of HER2 and Rb in of 23 patients represent the eligible population of the study and the absence of fulvestrant in patients with ER-positive and HER2- all had invasive cancer cells in tissue samples collected at week 2 positive (IHC 3+ score or gene ampliﬁcation by FISH) breast after beginning study therapy and at surgery. Table 1 describes cancers. In cohort C the triple block of HER2, ER and Rb was the main patient characteristics of the eligible population. Of note, studied in women with ER+ and HER2 tumors (ICH 1+ or 2+ 13% of the patients had clinical T3 or T4 and more than 50% had low score and not ampliﬁed). no clinically palpable axillary lymph nodes. 1 2 3 4 Fondazione Michelangelo, Milano, Italy. IEO, European Institute of Oncology, IRCCS, Milano, Italy. Azienda USL-IRCCS di Reggio Emilia, Reggio Emilia, Italy. Department of 5 6 Oncology, Azienda Sanitaria Universitaria Friuli Centrale, Udine, Italy. Addarii Medical Oncology IRCCS Azienda Ospedaliero-universitaria di Bologna, Bologna, Italy. IRCCS 7 8 Ospedale Policlinico San Martino, UO Breast Unit, Genova, Italy. Università di Genova, Dipartimento di Medicina Interna e Specialità Mediche (Di.M.I.), Genova, Italy. Department of Medical Oncology, San Raffaele Scientiﬁc Institute, Milano, Italy. Department of Oncology, Azienda Ospedaliero Universitaria di Ferrara - Arcispedale Sant’Anna, Ferrara, Italy. IRCCS European Institute of Oncology, Milano, University of Milan, School of Medicine, Milano, Italy. email: email@example.com Published in partnership with the Breast Cancer Research Foundation 1234567890():,; L. Gianni et al. Cohort B, HR+ HER2+, no fulvestrant Cohort C, HR+ HER2 low Total patients screened: 39 Total patients screened: 28 Not eligible 13 Not eligible 5 - HER2 negativity on retrospective assessment - HER2 positive on retrospective assessment at at referral laboratory 6 referral laboratory 1 - ER status ≤ 10% 2 - PgR status negative 2 - 2-week biopsy unavailable 1 - Ki67 < 20% 1 - Surgical biopsy unavailable 3 - Surgical biopsy unavailable 1 - Other therapy 1 Total eligible: 26 Total eligible: 23 Assessable for primary endpoint Ki67 changes Assessable for primary endpoint Ki67 changes • at week 2: 23 • at week 2: 25 of 26 • at surgery: 22 of 26 (pCR in 5 patients) • at surgery: 23 Assessed for secondary endpoints pCR and Assessed for secondary endpoints pCR and clinical objective response clinical objective response 26 patients 23 patients Assessed for safety profile Assessed for safety profile (received at least one cycle of therapy) (received at least one cycle of therapy) 39 patients 28 patients pCR: pathological complete response at surgery (absence of invasive cells) Fig. 1 CONSORT diagram. Cohort B (left) and Cohort C (right) report the total number of screened patients, the number of eligible patients for the primary and secondary endpoints, and the number of patients assessed for safety. week 2 the geometric mean went from 32.4 ± 19.9 to 2.6 ± 8.7 Table 1. Main patient characteristics. (P < 0.0001) and at time of surgery was 7.5 ± 20.4 (P < 0.001). Of interest, in 4 cases in the cohort B, Ki67 could not be measured Cohort B HR+ HER2+, Cohort C HR+ due to lack of tumor cells at surgery. In Table 2,we reportthe no fulvestrant HER2 low decrease of Ki67 at week 2 and at surgery below 10% that was Eligible patients 26 23 recently deﬁned as threshold for sensitivity to endocrine Median age in year 48.0 (29.0–86.0) 48.5 (35.0–79.0) therapy (ET) in tumors exposed to a brief course of ET ,and (range) at or below 2.7%, the threshold of complete cell cycle arrest T stage (CCCA) . In cohort B 64% of tumors had a drop below 10% at week 2 and 9.1% at surgery, while CCCA was reached by 36% cT1c 2 (7.7%) 2 (8.7%) at week 2 and none at surgery. In cohort C more than 90% cT2 19 (73.1%) 18 (78.3%) tumors showed a drop below 10% at week 2 and 56% at cT3 4 (15.4%) 3 (13.0%) surgery, while CCCA was achieved in 65% at week 2 and 30% at cT4b 1 (3.8%) – surgery (Table 2). The geometric mean for apoptosis went from Nodal status 1.0 ± 0.4 at baseline to 1.2 ± 0.4 at surgery in the 22 assessable cN0 15 (57.7%) 12 (52.2%) cases in Cohort B (P = 0.94) and from 1.0 ± 0.4 to 0.4 ± 0.4 in the 23 cases of cohort C (P = 0.058). cN1 11 (42.3%) 11 (47.8%) Histology Therapeutic activity Ductal invasive 26 (100%) 22 (95.7%) The therapeutic activity was analyzed during the entire duration of Lobular invasive 0 1 (4.3%) neoadjuvant therapy and at surgery. Clinical objective response PgR status immediately before surgery according to RECIST was reported in Positive 17 (65.4%) 23 (100%) 23 of 26 patients or 88.5% (95% CI 69.8–97.6) in cohort B and in 18 Negative = 0 5 (19.2%) 0 of 23 patients or 78.3% (95% CI 56.3–92.5) in cohort C. (Tables 3 HER2 + and 4). One patient in each cohort developed progressive disease while on treatment. At surgery, 5 of 26 patients (19.2%, 95% CI IHC 3+ 15 (57.7%) 6.6–39.4) had pCR in breast and nodes in cohort B, while no Neu ampliﬁed 11 (42.3%) patients in cohort C achieved a pCR. Effects on levels of KI67 Safety The primary goal of the trial was to study the effects of the The proﬁle of tolerability of the regimen was good. Overall, 2 drugs regimen on the levels of Ki67 at week 2 and/or at time of treatment-related serious adverse events were recorded in the surgery. As illustrated in Fig. 2, the decrease of Ki67 was highly study, one in cohort B (1 of 39 or 2.6%, 95% CI 0.1–13.5) and 1 in signiﬁcant at both time intervals. In cohort B, at week 2 the cohort C (1 of 28 or 3.6%, 95% CI 0.1–18.3). As reported in Tables 5 geometric mean (±SD, n = 25) went from 33.4 ± 12.7 to 5.5 ± and 6 for the two different cohorts, diarrhea was the most 12.4 (paired test P < 0·0001), and at time of surgery (n = 22) was frequent adverse event. Grade 3 toxic effects were infrequent and 25.7 ± 9.7 (P = 0.033). In cohort C, in all 23 eligible patients, at reported for diarrhea, stomatitis, and hypersensitivity reactions. npj Breast Cancer (2022) 1 Published in partnership with the Breast Cancer Research Foundation 1234567890():,; L. Gianni et al. Ki67 change from basal to week 2 to surgery Cohort C, HR+ HER2 Cohort B, HR+ HER2+, no fulvestrant low 100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 Fig. 2 Individual levels of Ki67 at baseline, week 2 and surgery. In cohort B, in 1 of 26 cases at week 2 the tumor block failed to reveal the presence of invasive cells and a pCR was reached at surgery. At surgery, residual invasive disease was present in 22 patients while 5 patients achieved a pathological complete remission with no invasive cells in the breast and axilla. In cohort C, all the 23 cases presented invasive cells at week 2 and at surgery. Table 2. Decrease of Ki67 at week 2 and at surgery. Table 3. Secondary endpoints: clinical response and pathological complete response rates in Cohort B (HER2+,ER+, no fulvestrant). Baseline (N = 26) Week 2 (N= 25) Surgery (N = 22) Eligible patients 26 COHORT B Mean (ranges) 37.1 (11–89) 11.2 (0–38) 27.8 (7–47) Overall clinical response 23 (88.5%, 95% CI 69.8–97.6) Drop below 10% 64% 9% Complete response 9 (34.6%, 95% CI 17.2–55.7) Drop to less than 2.7% 36% 0 Partial response 14 (53.8%, 95% CI 33.4–73.4) HER2 IHC 3+ (N= 15) (N = 14) (N = 13) Mean (ranges) 43.3 (25–89) 12.7 (0–38) 30.1 (9–47) Stable disease 2 (7.7%, 95% CI 0.9–25.1) Drop below 10% 57% 8% Progressive disease 1 (3.8%, 95% CI 0.1–19.6) Drop to less than 2.7% 35% 0 Pathological complete response 5 (19.2%, 95% CI 6.6–39.4) HER2 neu ampliﬁed (N= 11) (N = 11) (N = 9) (absence of invasive cells in breast Mean (ranges) 28.7 (11–44) 9.2 (0–29) 24.6 (7–36) and axilla) Drop below 10% 73% 11% HER2 IHC 3+ Drop to less than 2.7% 27% 0 Overall clinical response 13 (86.7%, 95% CI 59.6–98.3) Baseline (N = 23) Week 2 (N= 23) Surgery (N = 23) Complete response 6 (40.0%, 95% CI 16.3–67.7) COHORT C Partial response 7 (46.7%, 95% CI 21.3–73.4) Mean (ranges) 34.8 (21–78) 5.0 (0–34) 15.6 (1–87) Drop below 10% 91% 56.5% Stable disease 1 (6.7%, 95% CI 0.2–31.9) Drop to less than 2.7% 65% 30% Progressive disease 1 (6.7%, 95% CI 0.2–31.9) Pathological complete response 3 (20.0%, 95% CI 4.3–48.1) Neutropenia was the most frequent grade 3 event. Together with HER2 neu ampliﬁed neutropenia, stomatitis and diarrhea were the most frequent Overall clinical response 10 (90.9%, 95% CI 58.7–99.8) reasons for delay or discontinuation of palbociclib. No deaths Complete response 3 (27.3%, 95% CI 8.0–61.0) occurred during the study. Partial response 7 (63.6%, 95% CI 30.8–89.1) Stable disease 1 (9.1%, 95% CI 0.2–41.3) DISCUSSION Progressive disease 0 The extended study of two additional cohorts of the NA-PHER2 Pathological complete response 2 (18.2%, 95% CI 2.3–51.8) trial showed in cohort B of HER2-ampliﬁed breast carcinomas that CI conﬁdence interval. block of HER2 and cdk4/6 led to a rapid and major drop of Ki67 even without the use of estrogen receptor targeting in spite of estrogen receptor expression. We also showed that Ki67 rapidly and persistently dropped in cohort C of the NA-PHER2 trial, in response and 19.2% pCR. In cohort C the clinical response was which we applied the block of HER2, cdk4/6, and ER in women 78.3%, with 13% complete CR and no pCR. Tolerability and with HER2 breast cancer characterized by centrally conﬁrmed low feasibility of the multidrug regimens without and with fulvestrant ER and PgR positive immunohistochemistry, and HER2 expression were good without observation of any major or limiting toxicity, as at 1+/2+ without gene ampliﬁcation. In cohort B the block of already described for cohort A of the study . HER2 and cdk4/6 without fulvestrant was associated with an A vast literature supports the value of Ki67 as a marker of 88.5% rate of objective response, 34.6% of complete clinical potential sensitivity to endocrine therapy of HR+ breast Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2022) 1 Ki67 L. Gianni et al. Table 4. Secondary endpoints: clinical response and pathological Table 6. Selected treatment emergent adverse events (TEAE, Safety complete response rates in Cohort C (HR+ HER2 ). population = 28 patients) in Cohort C (HR+ HER2 ). low low Eligible patients 23 % Any TEAE (95% CI) % Grade 3 TEAE (95% CI) Overall clinical response 18 (78.3%, 95% CI 56.3–92.5) Diarrhea 53.6 (33.9−72.5) 7.1 (0.9−23.5) Complete response 3 (13.0%, 95% CI 2.8–33.6) Neutropenia 50.0 (30.6−69.4) 42.9 (24.5−62.8) Partial response 15 (65.2%, 95% CI 42.7–83.6) Stomatitis 28.6 (13.2−48.7) 3.6 (0.1−18.3) Stable disease 4 (17.4%, 95% CI 5.0–38.8) Mucosal 25.0 (10.7−44.9) 7.1 (0.9−23.5) Progressive disease 1 (4.3%, 95% CI 0.1–21.9) inﬂammation Pathological complete response 0 Pyrexia 25.0 (10.7−44.9) − (absence of invasive cells in breast and axilla) Aphthous ulcer 14.3 (4.0−32.7) − Headache 14.3 (4.0−32.7) − CI conﬁdence interval. Asthenia 10.7 (2.3−28.2) − Fatigue 10.7 (2.3−28.2) − Nausea 10.7 (2.3−28.2) − Table 5. Selected treatment emergent adverse events (TEAE, Safety Leukopenia 10.7 (2.3−28.2) 3.6 (0.1−18.3) population = 39 patients) in Cohort B (HR+ HER2+, no fulvestrant). Arthralgia 10.7 (2.3−28.2) − % Any TEAE (95% CI) % Grade 3 TEAE Dyspepsia 10.7 (2.3−28.2) − (95% CI) Hot ﬂush 10.7 (2.3−28.2) − Neutropenia 64.1 (47.2−78.8) 30.8 (17.0−47.6) Hypertension 10.7 (2.3−28.2) − Diarrhea 59.0 (42.1−74.4) 7.7 (1.6−20.9) Anemia 7.1 (0.9−23.5) − Mucosal inﬂammation 33.3 (19.1−50.2) − ALT increased 7.1 (0.9−23.5) − Pyrexia 28.2 (15.0−44.9) − AST increased 7.1 (0.9−23.5) 3.6 (0.1−18.3) Stomatitis 28.2 (15.0−44.9) 5.1 (0.6−17.3) LVEF decrease 3.6 (0.1−18.3) − Asthenia 20.5 (9.3−36.5) − CI conﬁdence interval. Fatigue 20.5 (9.3−36.5) − Nausea 17.9 (7.5−33.5) − Epistaxis 17.9 (7.5−33.5) − for sensitivity to ET at day 15 set in the POETIC trial . Of note, at Rash 17.9 (7.5−33.5) − week 2 nine of the patients in cohort B (36%) had a drop of Ki67 to Vomiting 15.4 (5.9−30.5) − less than 2.7%, the threshold for Complete Cell Cycle Arrest (CCCA) Anemia 12.8 (4.3−27.4) − that is a more stringent threshold indicating sensitivity to Leukopenia 12.8 (4.3−27.4) 5.1 (0.6−17.3) treatment in ER+ tumors . The lack of endocrine therapy in the therapeutic regimen in cohort B of the NA-PHER2 may question Folliculitis 10.3 (2.9−24.2) − the predictive relevance and the actual meaning of the Ki67 drop Hemorrhoids 10.3 (2.9−24.2) − at week 2. However, the rate of CCCA is of the same order of ALT increased 7.7 (1.6−20.9) 2.6 (0.1−13.5) magnitude of that recently reported for the cell cycle inhibitor AST 7.7 (1.6−20.9) − abemaciclib without ET in women with ER+ breast cancer enrolled in the neo-MONARCH study . Importantly, very high values of CI conﬁdence interval. Ki67 were present at the baseline core biopsy in the cases reported here, with a mean value of 37%. This is in line with the ﬁndings in the POETIC trial of consistently higher values of Ki67 in carcinoma. Recently, the decrease of Ki67 at week 2 after a brief HER2+ than HER2− breast cancer cases . Overall, the relevant course of neoadjuvant perioperative endocrine therapy was rate of objective clinical response and the observation of validated in the large POETIC trial as predictor of long-term pathologic complete responses is suggestive of a possible beneﬁt of endocrine therapy in women with ER+ operable breast association between observed therapeutic efﬁcacy and extent of cancer . A drop of Ki67 > 20% at day 15 of therapy with Ki67 modulation in cohort B. However, the lack of endocrine anastrozole was used in HER2+ tumors as reference to continue therapy in the regimen and the use of palbociclib that blocks cell therapy with endocrine treatment and HER2 block in the PerElisa cycle progression deﬁnes the link between Ki67 changes and trial . In the ﬁrst cohort of NA-PHER2 we had shown a rapid, prediction of treatment sensitivity only as a reasonable hypothesis signiﬁcant and very large drop of Ki67 at week 2 during that requires appropriate test in a prospective validation trial with neoadjuvant therapy with ET and block of HER2 and of cdk4/6 . event free survival as endpoint. A similar large effect on Ki67 was conﬁrmed in cohort B of the As already observed in cohort A of the study ,valuesofKi67 study in ER+ and HER2+ tumors even without direct targeting of in cohort B tended to rebound at time of surgery even though the estrogen receptor. The decision of testing the effects of dual remaining lower than at baseline. A similar trend was already block of HER2 and inhibition of cdk4/6 without ET was based on reported in other neoadjuvant trials of cdk 4/6 inhibitors 13–16 18,19 the well-known relative resistance of HER2+ tumors to ET , (neoPalAna e neoMONARCH) . In neoMONARCH the rebound and the demonstration that there exists a preferential sensitivity was apparently linked to discontinuation of treatment with to palbociclib monotherapy in ER+ and HER2-ampliﬁed cell lines abemaciclib and anastrozole for more than 4 days .In our in the absence of ER targeting . At week 2 all patients enrolled study treatment was discontinuedfor avariableintervalfrom into cohort B had a decrease of Ki67, and 64% of them had a drop end of planned therapy and time of surgery. In cohort B the below 10%, the value that was recently validated as the threshold rebound effect was more frequent and more pronounced npj Breast Cancer (2022) 1 Published in partnership with the Breast Cancer Research Foundation L. Gianni et al. than that reported in cohort A but also in cohort B we were enrolled in the ETNA trial and exposed to a 16 weeks-long unable to measure an effect of duration of drug-free interval on chemotherapy with taxanes and anthracyclines had a rate of the rebound. clinical response similar to that observed in the present cohort The dramatic drop of Ki67 at week 2 and the persisting drop at and a rate of pCR of only 10% . time of surgery were not associated with increased apoptosis, Our study has obvious limitations. The number of cases is small which was assessed at the same time intervals as Ki67. The data on and limits the interpretation of the ﬁndings, and the therapeutic apoptosis suggests that also in those cases in whom excellent results were limited to clinical and pathological response without clinical response was observed the efﬁcacy was due to alternative additional follow up for disease free survival after surgery. In mechanism of tumor shrinkage, including senescence which is a addition, changes of Ki67 values during and after treatment as well-known mechanism of biologic effects of cdk4/6 inhibitors . predictor of sensitivity were only validated for endocrine therapy In the present study we also showed that Ki67 rapidly and and could have no such predictive value in the context of the persistently dropped in cohort C of the NA-PHER2 trial, in which regimens tested in the trial. However, the ﬁndings are consistent we applied the block of HER2, cdk4/6, and ER in women with with the assumption that it may be worth testing the neoadjuvant HER2 breast cancer that was characterized by ER and PR application of a chemotherapy free approach to women with low positive immunohistochemistry, and HER2 expression at 1+/2+ early breast cancer and HER2+ tumors while investigating in without gene ampliﬁcation, as assessed at a central laboratory. Per more detail the relative role and need for a pharmacologic block protocol entry criteria, none of the patients in cohort C had a of the estrogen receptor, the cell cycle, the erbB-family of tumor with starting Ki67 below 20% at baseline. Interestingly, the receptors and achieve an effective therapy tailored to the drop of Ki67 was more persistent and profound than in cohort B individual characteristics of the tumor. To move closer to such and rebound was less frequent. Ki67 had values lower than 10% in goal we are actively studying the molecular characteristics of the 91.3% and 56.5.1% at week 2 and at surgery, respectively, and a serial collection of tumors and blood of the NA-PHER2 trial. Such Ki67 in the range of CCCA in 65.2% of cases at week 2 and 30.4% ongoing ancillary investigation may better inform the design of at surgery. The data in Ki67 changes are similar to those reported clinical trials to test the real value of the chemo-free regimens for abemaciclib and anastrozole in the neoMONARCH study explored in the NA-PHER2 study. although the rebound effect is again less pronounced and not linked to the duration of discontinuation of therapy until surgery. METHODS Also in cases with HER2 tumors apoptosis was not apparently low involved in the mechanism of observed therapeutic activity. Study design and patients Overall, the reported additional cohorts of the NA-PHER2 study NA-PHER2 was an open-label, multicenter, exploratory phase II study illustrate the course of Ki67 during a 16-weeks long therapy performed in 7 Italian sites. The original protocol included only a cohort of targeting cdk4/6 and HER2, and provide potentially useful patients (Cohort A) who received a 4-drug regimen named HPPF which included trastuzumab, pertuzumab, palbociclib, and fulvestrant. A protocol information for the ongoing effort of deﬁning novel and amendment included two additional cohorts of patients, named cohort B chemo-free treatments for women with HER2 positive breast and cohort C. Most of the criteria and methods used in the study were cancer and hormone receptor positive breast cancer. The already llustrated . standard approach to date is based on targeting the HER2 In the section of the present report we will detail features that are receptor in combination with chemotherapy irrespective of the characteristic of the two additional cohorts of the trial. The primary aim estrogen and progesterone receptor status of the tumor. In was to characterize, separately for each cohort, Ki67 changes from baseline Cohort B of the study as in the previously reported Cohort A before therapy at 2 weeks after starting therapy, and at surgery. Apoptosis there is a relevant rate of Ki67 drop at week 2 that would qualify changes were also assessed from baseline to surgery. these cases as responsive if they were exposed to ET only as in Secondary aims were: rate of pathological complete response (pCR) the POETIC trial . The course of Ki67 is accompanied by deﬁned as absence of invasive cells in breast and axilla at surgery; clinical excellent clinical and even pathological response. Also the rate objective response rate at the end before surgery; and safety proﬁle of the experimental therapy. of progressive disease is in line with literature data focusing on Patients eligibility required presence of previously untreated histologi- neoadjuvant chemotherapy. The number of cases treated in the cally conﬁrmed unilateral invasive, HER2-positive (for cohort B) or HER2 low current study is small and exploratory, but the rate of objective (for cohort C) and ER-positive (>10%) breast cancer. HER2 positive disease response and of pCR is of the same order of magnitude than that was always assessed according to ASCO/CAP guidelines in use at the time reported for ER+ tumors exposed to dual block of HER2 and of the centralized review. Additional eligibility criteria for enrolment in chemotherapy in the NeoSphere trial . We think that the cohort C were positive progesterone receptor (PgR) status and Ki67 > 20%. chemotherapy-free approach described in the NA-PHER2 Patients had to consent to provide tumor tissues for centralized deserves additional attention also in the light of the recent conﬁrmation of HER2 and ER status and assessment of Ki67 values at report of the KATHERINE trial that has documented the beneﬁtof required times. Other required characteristics were: age 18 years or older; ECOG performance status ≤ 1; tumor stage classiﬁed as cT1c to cT4a-d. Key switching HER2-directed treatment from naked antibodies exclusion criteria were metastatic disease, bilateral breast cancer, other (mostly trastuzumab, but also trastuzumab and pertuzumab) to malignancies, inadequate bone marrow or renal function, impaired liver T-DM1 in cases of residual disease after neoadjuvant therapy . function, impaired cardiac function, uncontrolled hypertension, pregnancy, Of note, the majority of patients with residual disease who and refusal to use contraception. HER2 status, ER, PgR and Ki67 had to be entered the KATHERINE study had hormone receptor positive conﬁrmed centrally. disease . The observation is not only illustrating once again that The study was conducted according to Good Clinical Practice guidelines HER2 and ER-positive breast cancers have a differential sensitivity and the Declaration of Helsinki. All patients provided written informed to treatment with anti-HER2 therapy combined with chemother- consent. Approvals for the study protocol (and any modiﬁcations thereof) apy, but also that such tumors deserve investigation of a more were obtained from independent ethics committees at each participating institution and relevant competent authority. targeted approach that may avoid unnecessary toxicity. The Cohort C in cases with HER2 breast cancer deserves low additional considerations. Patients were selected to have Treatment and procedures hormone receptors positive tumors including the expression of Within 5 days from registration, patients had to start protocol treatment, progesterone receptor. They also all had to have a Ki67 value of consisting of HPP for cohort B and HPPF for cohort C. Trastuzumab (H) at least 20%. In brief, the patients were selected to have a was given at 8 mg/kg loading dose IV on cycle 1, and then at 6 mg/kg IV luminal-B like type of breast cancer. It is of interest that a every 3 weeks. Pertuzumab (P) at 840 mg loading dose IV at cycle 1, and similarly selected group of patients with luminal B-like tumors then at 420 mg IV every 3 weeks. H and P were given for total Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2022) 1 L. Gianni et al. 6 administrations. Palbociclib (P) was given at 125 mg po q.d. for 21 days deﬁned by modiﬁed RECIST criteria previously described . The pathological followed by one week rest (1 cycle to covering 4 weeks) and repeated for complete response was deﬁned as the absence of invasive disease in total 5 cycles. In cohort C fulvestrant (F) was given intra-muscle at 500 mg breast and axillary nodes at surgery. Exact 95% conﬁdence intervals every 4 weeks and repeated for 5 times, with an additional 500 mg dose (Clopper–Pearson method) were calculated for clinical response and given 2 weeks after the ﬁrst one. The total duration of neoadjuvant pathological complete response rates. palbociclib (5 cycles every 4 weeks) and fulvestrant (5 administrations Toxicity reported adverse events were categorized according to the NCI- every 4 weeks plus the additional dose) was selected to match as closely CTCAE version 4.0. Treatment-emergent adverse events (TEAE) were assessed by clinical examination, questioning for symptoms of toxicity, as possible the total duration of the six 3-weekly planned administrations laboratory assessments (hematology and biochemistry), vital signs, at each of H and P. Premenopausal women received an LHRH-agonist. physical examination during neoadjuvant therapy, before surgery and In presence of severe toxicity it was up to the investigators to decide for within 4–5 weeks after surgery. Cardiac examination with ECG and LVEF treatment discontinuation and immediate surgery. During neoadjuvant were to be repeated after the third cycle of neoadjuvant therapy and therapy physical, hematological and biochemical exams were performed before surgery. Patients were analyzed according to the worst grade before each treatment cycle and repeated before surgery. Clinical response reported throughout the whole treatment period. TEAEs were assessed for in breast and nodes was assessed before surgery and was deﬁned by all patients who received at least one treatment cycles, including patients modiﬁed RECIST criteria. Toxicity reported adverse events were categor- who retrospectively failed to meet all eligibility criteria. ized according to the NCI-CTCAE version 4.0. After neoadjuvant therapy No sensitivity or interim analyses were planned. We analyzed data with patients underwent surgery and pCR was assessed by local pathologist SAS statistical software (version 9.4). according protocol guidelines. Following surgery, additional adjuvant systemic therapy was given including chemotherapy (especially for patients with high tumor Reporting summary burden at baseline, positive axillary nodes at surgery, and at the Further information on research design is available in the Nature Research Investigator’s discretion) plus standard HER2 treatment until comple- Reporting Summary linked to this article. tion of full 1yearif HER23+ or neu ampliﬁed and endocrine therapy according to local guidelines. Post-surgery irradiation was recommended in all patients after breast- DATA AVAILABILITY conserving surgery and in selected patients who underwent mastectomy The datasets generated during and/or analyzed during the current study are available according to international and local guidelines. from the corresponding author on reasonable request. Assessments Received: 15 June 2021; Accepted: 6 December 2021; The primary endpoint was Ki67 expression level changes from baseline. Ki67 was scored in the central laboratory and reported in eCRF as the percentage of positively staining cells within the invasive margin in the examined area. Manual (visual) counting was performed, and percent of immunostained invasive tumor cells was assessed out of 500 cells or out of all the invasive tumor cells in the sections if less than 500 cells were REFERENCES present. A co-primary endpoint was to assess the changes in apoptosis 1. Witzel, I. I., Koh, L. F. & Perkins, N. D. Regulation of cyclin D1 gene expression. from baseline to surgery. Changes in apoptosis were evaluated by Biochem Soc. Trans. 38, 217–222 (2010). counting apoptotic bodies at baseline and at surgery. Tumor samples 2. Prall, O. W., Rogan, E. M. & Sutherland, R. L. Estrogen regulation of cell cycle were collected locally at all speciﬁed time points in all patients and stored progression in breast cancer cells. J. Steroid Biochem. Mol. 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The role of hormonal therapy in the management of COMPETING INTERESTS hormonal-receptor-positive breast cancer with co-expression of HER2. Nat. Clin. L.G. has been an Advisory Board member and/or consultant for ADC Therapeutics, Pr. Oncol. 5, 531–542 (2008). AstraZeneca, Celgene, Eli Lilly, G1 Therapeutics, Genentech, Genomic Health, Merck 14. Houston, S. J. et al. Overexpression of c-erbB2 is an independent marker of Sharp & Dohme, Oncolytics Biotech, Odonate Therapeutics, Onkaido Therapeutics, resistance to endocrine therapy in advanced breast cancer. Br. J. Cancer 79, Roche, Pﬁzer, Taiho Pharmaceutical, Hexal Sandoz, Seattle Genetics, Synthon, 1220–1226 (1999). Zymeworks, Sanoﬁ-Aventis, Forty Seven (CD47), GENENTA, METIS Precision Medicine, 15. Arpino, G., Wiechmann, L., Osborne, C. & Schiff, R. Crosstalk between the estrogen Synafﬁx, Menarini Ricerche, Amgen, Biomedical Insights Inc., and a Research Grant receptor and the HER tyrosine kinase receptor family: molecular mechanism and from Revolution Medicines to his institution outside the submitted work. M.C. has clinical implications for endocrine therapy resistance. Endocr. Rev. 29, 217–233 been an Advisory Board member for AstraZeneca, Pierre Fabre, Pﬁzer, OBI Pharma, (2008). Puma Biotechnology and Celldex, received honoraria from Novartis, and Research 16. Giuliano, M. et al. Upregulation of ER Signaling as an adaptive mechanism of cell Grant from Roche to his institution outside the submitted work. M.M. reports personal survival in HER2-positive breast tumors treated with anti-HER2 therapy. Clin. fees from Novartis, Roche, Celgene, Eisai, AstraZeneca, Pﬁzer, Eli Lilly and MSD Italia Cancer Res. 21, 3995–4003 (2015). outside the submitted work. C.Z. has been an Advisory Board member for Roche, 17. Finn, R. S. et al. Efﬁcacy and safety of palbociclib in combination with letrozole as Eisai, Novartis, AstraZeneca, Pﬁzer, PharmaMar, Amgen, Tesaro, Lilly, Celgene, ﬁrst-line treatment of ER-positive, HER2-negative, advanced breast cancer: personal fee from Pierre Fabre and Istituto Gentili, and Research Grants from Roche, expanded analyses of subgroups from the randomized pivotal trial PALOMA-1/ Novartis, AstraZeneca, Pﬁzer, Seattle Genetics, Tesaro, Pierre Fabre, Istituto Gentili, TRIO. Breast Cancer Res. 18,67–81 (2016). Takeda, TEVA, Medivation, AbbVie, Array BioPharma, Morphotek and Synthon to his 18. Hurvitz, S. A. et al. Potent Cell-Cycle inhibition and upregulation of immune institution outside the submitted work. L.D.M. reports personal fees from Roche, response with abemaciclib and anastrozole in neoMONARCH, Phase II neoadju- Novartis, Pﬁzer, Ipsen, Takeda, Eli Lilly, Amgen, Daiichi Sankyo, Seagen, Genomic + - vant study in HR /HER2 Breast Cancer. Clin. Cancer Res. 26, 566–580 (2020). Health, Exact sciences, MSD, AstraZeneca and non-ﬁnancial support from Celgene, 19. Ma, C. X. et al. NeoPalAna: Neoadjuvant palbociclib, a cyclin-dependent kinase Pﬁzer, Roche, outside the submitted work. G.B. has been an Advisory Board, 4/6 inhibitor, and anastrozole for clinical stage 2 or 3 estrogen receptor-positive consultant or received personal fees from Roche, Pﬁzer, AstraZeneca, Lilly, Novartis, breast cancer. Clin. Cancer Res. 23, 4055–4065 (2017). Neopharm, Amgen, MSD, Chugai, Sanoﬁ, Daiichi Sankyo, EISAI, Genomic Health. A.F. 20. Klein, M. E., Kovatcheva, M., Davis, L. E., Tap, W. D. & Koff, A. CDK4/6 Inhibitors: the reports personal fees and non-ﬁnancial support from Roche, Novartis, AstraZeneca, mechanism of action may not be as simple as once thought. Cancer Cell 34,9–20 Pﬁzer, Eli Lilly, Daichii and Seagen outside the submitted work. G.V. reports personal (2018). fees from Roche, MSD, BMS, AstraZeneca, Celgene, Dako and personal fees da Daichii 21. von Minckwitz, G. et al. Trastuzumab emtansine for residual invasive HER2- Sankyo outside the submitted work. The other authors have no conﬂicts of interest positive breast cancer. N. Engl. J. Med. 380, 617–628 (2019). to declare. 22. Gianni, L. et al. Comparing neoadjuvant nab-paclitaxel vs paclitaxel both followed by anthracycline regimens in women with ERBB2/HER2-negative breast cancer- the evaluating treatment with neoadjuvant abraxane (ETNA) trial: a randomized ADDITIONAL INFORMATION phase 3 clinical trial. JAMA Oncol. 4, 302–308 (2018). Supplementary information The online version contains supplementary material 23. Dowsett, M. et al. Assessment of Ki67 in breast cancer: recommendations from available at https://doi.org/10.1038/s41523-021-00377-8. the international Ki67 in breast cancer working group. J. Natl Cancer Inst. 103, 1656–1664 (2011). Correspondence and requests for materials should be addressed to Luca Gianni. 24. Pathmanathan, N. & Balleine, R. L. Ki 67 and proliferation in breast cancer. J. Clin. Pathol. 66, 512–516 (2013). Reprints and permission information is available at http://www.nature.com/ reprints ACKNOWLEDGEMENTS Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims We are indebted to all the patients who have participated in our clinical trial and to in published maps and institutional afﬁliations. the many associates, in particular medical oncologists, surgeons, radiation therapists, pathologists, research nurses, and data managers, for their cooperation during the study. We are particularly indebted to Raffaella De Fato and Simona Barlera, former biostatisticians, and to Nadia Malinverni, Clinical Study Coordinator, at Michelangelo. Open Access This article is licensed under a Creative Commons The study was supported by an unrestricted grant from Pﬁzer Italia S.r.l. and from Attribution 4.0 International License, which permits use, sharing, Roche S.p.a. Italy. This work has been also supported in part by a Breast Cancer adaptation, distribution and reproduction in any medium or format, as long as you give Research Foundation grant to Luca Gianni. appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless AUTHOR CONTRIBUTIONS indicated otherwise in a credit line to the material. If material is not included in the The study was designed by L.G. and P.V. in collaboration with the sponsor, article’s Creative Commons license and your intended use is not permitted by statutory Fondazione Michelangelo. Data were collected via an electronic data capture system regulation or exceeds the permitted use, you will need to obtain permission directly located in Milano and analyzed by the biostatistician I.M. G.V. assessed all biomarkers from the copyright holder. To view a copy of this license, visit http://creativecommons. required for the study. L.G. and P.V. prepared the initial draft of the manuscript. org/licenses/by/4.0/. All authors made substantial contributions to the intellectual content of this work via data acquisition and interpretation, in addition to critical revision of the manuscript. All authors provided their ﬁnal approval of the manuscript. © The Author(s) 2022 Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2022) 1
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